The use of fluorescent protein tags has revolutionized all aspects of cell biology research during the last 15 years, enabling analysis of the dynamic structures within living cells. The cloning of Green Fluorescent Protein (GFP) and the subsequent development of additional fluorescent proteins with a wide range of spectral properties made this revolution possible. The newest versions of fluorescent proteins are photo-activatable (PA), allowing higher dynamic and spatial resolution of proteins in cells. The dynamic behavior of tagged proteins can be examined by local activation of molecules, and observation of protein movement over time in live tissue. PA-proteins have also enabled new methods of super-resolution fluorescence microscopy that yield composite images with sub-100 nm resolution, breaking the diffraction limit of light microscopy. The goal of this proposal is to significantly expand the experimental applications of PA proteins for imaging dynamic cellular processes and super-resolution fluorescence microscopy in intact tissues. To achieve this goal, we will engineer PA tags into Drosophila genes in their genomic context to allow analysis of proteins expressed at their endogenous levels under native transcriptional control. We will use these PA-tagged proteins to develop super-resolution imaging of intact ovarian egg chambers that provide a beautifully graded set of challenges for super-resolution microscopy - germline ring canals of gradually increasing size found within gradually thicker egg chambers, and much smaller ring canals within epithelial cells surrounding egg chambers. Our specific aims are: 1) produce Drosophila stocks expressing PA-tagged proteins at endogenous levels using recombineering in newly available BAC clones of Drosophila genomic DNA;2) develop super-resolution imaging for analysis of ring canals using biplane fluorescence photoactivation localization microscopy (BP FPALM) of whole-mount Drosophila egg chambers;3) characterize protein dynamics in living ring canals using 2-photon activation and confocal microscopy of PA-tagged ring canal proteins. This project will impact the entire cell biology community by dramatically expanding the applications of nascent super-resolution technology. Investigations of cell biology in experimental systems such as Drosophila, zebrafish, mouse and C. elegans that have extensive genetic tools and robust genome projects will find the tools generated by this project especially useful. PUBLIC HEALTH RELEVANCE: The ability to observe the inner workings of cells is crucial for understanding function in both normal conditions and disease processes. New methods for super-resolution microscopy have emerged in the last few years that dramatically improve analysis of cell biology. This project will significantly expand the use of super-resolution microscopy to the study of a wide range of proteins in intact animal tissues, which will enable much more detailed analysis of the cell biology in normal and diseased tissue.